CN109320625B - 一种当归多糖的提取方法及应用 - Google Patents
一种当归多糖的提取方法及应用 Download PDFInfo
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Abstract
本发明属于医药技术领域,具体涉及一种当归多糖的提取方法及其应用。试验结果显示,当归多糖可以显著降低辐射引起的大鼠血清DAO含量升高,可显著减轻辐射引起的大鼠肠壁的损伤。当归多糖可以显著降低辐射引起的大鼠有核细胞、白细胞含量降低,可显著减轻辐射引起的大鼠造血功能的减退。当归多糖可以显著降低辐射引起的大鼠大便球杆比含量升高,可显著减轻辐射引起的大鼠肠道菌群失调。
Description
技术领域
本发明属于医药技术领域,具体涉及一种当归多糖的提取方法及其应用。
背景技术
随着各类高科技产品的应运而生,核医学、放射医学诊疗技术的不断提高及广泛应用,人们生活环境充满了各类辐射源,辐射对机体影响非常之大,防治急性放射性损伤引起的并发症,是我们临床上亟待解决的难题,肠道是辐射损伤的敏感器官之一,因此对辐射诱发肠道损伤的预防及其机制研究具有十分重要的现实意义。现代研究发现当归多糖具有改善免疫功能的作用,当归多糖主要组分有改善肠道菌群的作用。
人们常说病从口入,肠粘膜免疫是肠屏障的关键环节,它由肠相关淋巴组织(gut-associated lymphoid tissues,简称GALT)构成。在肠相关淋巴组织中,上皮内淋巴细胞(intraepithelial lympho-cyte,简称IEL)是整个免疫系统中与肠腔抗原最接近的免疫细胞,同时上皮内淋巴细胞和上皮细胞毗邻,与肠上皮细胞间存在着双向免疫调节,因此上皮内淋巴细胞在肠粘膜免疫中发挥着独特而的作用。包括放射性损伤在内的多种致病因素都可能造成肠粘膜免疫屏障的损伤 ,使机体对外来抗原和病原体的敏感性增强 ,引起肠源性感染等疾病的发生 ,加重致伤效应或延迟恢复过程。
每个成年人机体皮肤粘膜上的微生物总量超过1×1014个,是人体自身细胞数目的10倍。现已得知微生物是人体生长发育必不可少的元素,表现在营养物质的吸收和免疫功能的稳定等方面。目前认为人体有两大基因组,一是来自父母的基因组,另一个是在肠道共生的微生物基因组,被称为“元基因组”。有研究表明人类转录组的 4026600 个m RNA 中约有 4×106个 m RNA来源于肠道菌群, 从人出生就逐步开始的这种基因层面上的交流在功能上体现为肠道菌群广泛参与人体的各项生理活动,如饮食物的消化和吸收、神经的发育以及免疫等等。肠道菌群作为人体重要的微生态系统之一,具有增强人体免疫功能的作用。肠道菌群和肠粘膜屏障相互作用,相互协调,共同维护着机体的生理功能。
现代免疫学和医学微生态学认为,疾病的病变过程反映机体免疫系统对外来的抗原和自身抗原的免疫激活、应答等一系列免疫反应过程。人体的感染是病原微生物引起的人体异常反应。其是否发病不仅取决于病原微生物自身的属性,更取决于人体的微生态平衡以及这种平衡状态对人体免疫系统的激活及其应答。肠道微生物具有免疫增强功能,正常微生物群作为抗原物质,先是非特异性地促进机体免疫器官发育成熟,并且特异性地持续刺激机体免疫系统发生免疫应答,进而机体所产生的免疫物质能对具有交叉抗原组分的病原菌有某种程度的抑制或杀灭作用。Liang S等研究发现,菌群失调会使宿主的脾细胞增殖功能及IL-1和IL-2的活性明显降低。张亚超等研究发现肠道菌群失调时,骨髓中粒细胞的数量减少,同时外周血和肝脏中的粒细胞数量也会降低,所以肠道菌群与机体的免疫功能相互作用,共同维护着机体的生理功能,
双歧杆菌(Bifidobacterium)是1899年由法国学者Tissier从母乳营养儿的粪便中分离出的一种厌氧的革兰氏阳性杆菌,末端常分叉,故名双歧杆菌。它是存在于人体内的一种生理细菌,是人体益生菌中最重要的一大类。许多关于肠道微生态的研究表明,双歧杆菌在婴儿肠道微环境菌群中占有主导地位,同时也是成人肠道微环境菌群中的优势菌之一。目前经研究证实能够促进人体健康的双歧杆菌主要有:两歧双歧杆菌、婴儿双歧菌、长双歧杆菌、短双歧杆菌、青春双歧杆菌、角双歧杆菌、链状双歧杆菌、假链状歧杆菌等。双歧杆菌的有益作用毋庸置疑,但也有双歧杆菌引起不良反应及产生耐药性的相关报道。
双歧杆菌在维持肠道微环境平衡、抑制病原菌群生长等方面发挥着重要作用,双歧杆菌依靠其强大的粘附和定植能力竞争性粘附于肠道上皮细胞达到拮抗致病菌的作用,双歧杆菌还可合成一些酸性物质调节肠道pH来抑制病原菌的生长。来自浙江大学动物分子营养学教育部重点实验室的研究证实两歧双歧杆菌对大肠杆菌K88具有拮抗作用。其抑菌机制主要是由于双歧杆菌在代谢过程中的主要产物有机酸,如乙酸和乳酸等,导致培养液中的pH值和氧化还原电位Eh降低,造成不利于病原菌生长的环境,在一定程度上可以降低病原菌的代谢速率,最终抑制其生长。有相关报道表明通过对婴儿双歧杆菌发酵液对致病菌生长抑制作用的研究,得出了婴儿双歧杆菌对金黄色葡萄球菌、绿脓杆菌、甲型副伤寒杆菌、乙型副伤寒杆菌、福氏痢疾杆菌、埃希氏大肠杆菌、白色念珠菌、变形杆菌的生长繁殖有明显的抑制作用。婴儿双歧杆菌对致病菌生长的抑制作用,一是因为产生有机酸,使环境pH值下降,二是由于产生抗菌物质。许多临床研究也证实了双歧杆菌对慢性腹泻及儿童抗生素相关性腹泻有明确的疗效。研究表明双歧杆菌对肠道微环境调控作用具有较好疗效,临床上在肠道微环境失衡方面的应用值得推广。
肠道菌群结构的变化直接影响和决定着人体的健康状况,研究肠道菌群的组成变化可以从一个侧面整体上反映机体的生理功能状况。中医药对肠道微生态的调节研究日益受关注,不仅从理论研究,还是临床和动物实验研究,都已证实中药有助于维持肠道微生态的平衡,调节肠道菌群。
当归多糖的主要成分有D-葡萄糖(D-glucose)、鼠李糖、D-半乳糖、以阿拉伯糖(Larabinose)、甘露糖(mannose)、岩藻糖(focose)、D-木糖(D-xylose) 及葡萄糖醒酸(glucuronic acid)和半现糖醋酸(galacturonicaciD)。
当归多糖有多种作用。在血液系统,当归多糖能增加外周血红细胞、白细胞、血红蛋白及骨髓有核细胞数,并且这种作用在外周血细胞减少和骨髓受到抑制时尤为明显。有免疫促进作用,当归多糖不仅能激活T、B淋巴细胞、巨噬细胞等免疫细胞,还能促进细胞因子生成,促进抗体生成。当归多糖还有抗肿瘤作用、抗氧化作用、保肝作用、抗辐射损伤等作用,其中抗辐射作用主要体现在改善免疫和血液系统方面。
文献孙元琳《当归多糖的制备结构分析和抗辐射效应研究》中公开了当归多糖的抗辐射作用具体为:体外细胞培养试验结果表明,当归多糖对辐射引发的肝细胞增殖活性的抑制有良好的防护作用。能有效地抑制骨髓PCE微核的形成,加速造血机能的恢复,降低外周血淋巴细胞的凋亡水平,使外周血白细胞和淋巴细胞数量回升;同时通过促进外周血淋巴细胞转化、降低肝细胞的膜脂质过氧化水平,增强机体的辐射耐受性, 提示当归多糖具有一定的抗辐射功能,对亚急性辐射损伤小鼠有良好的防护作用。体内实验考察了当归多糖对亚慢性辐射损伤小鼠的防护作用。结果表明,当归多糖能有效地抑制骨髓细胞凋亡的发生和骨髓PCE微核的形成,加速造血机能的恢复,使外周血白细胞数量回升;同时通过促进脾淋巴细胞转化和肝细胞增殖活性、抑制肝细胞凋亡、降低睾丸细胞的膜脂质过氧化水平,保护生殖系统功能来增强机体的辐射耐受性,提示当归多糖具有一定的抗辐射功能,对亚慢性辐射损伤小鼠有良好的防护作用。
当归多糖可以有效抑制辐射损伤引发的骨髓造血细胞的凋亡。关雪晶等研究结果表明,当归多糖能提高辐射损伤小鼠骨髓基质细胞的贴壁和增殖能力,加速骨髓基质细胞周期转换和降低凋亡率,增强骨髓基质细胞表面黏附分子 CD54 和 CD106 的表达,改善骨髓造血微环境,进而促进放射损伤后造血功能恢复。谢丛华等开展雌性小鼠实验,对成年雌性小鼠腹腔内注射当归注射液,结果表明当归能降低该过程中TNF-α表达水平,可能通过调控细胞因子起到防辐射作用。
但是,上述文献中仅公开了当归多糖对辐射引起的肝功能、免疫系统和生殖系统的影响,未见对辐射所致肠道屏障损伤的报道。
发明内容
本发明的目的在于提供一种当归多糖的提取方法。
发明的另一目的在于提供当归多糖的新用途。
本发明是通过下述的技术方案来实现的:
一种当归多糖的提取方法,所述当归多糖提取方法为:将自然风干后的新鲜当归,直接用蒸馏水煮沸30min,料液比为1∶30,提取液用无水乙醇调乙醇终浓度至80%,搅拌均匀后,4℃冷藏静置6h后离心,沉淀用95%%乙醇去除色素直到无色为止,沉淀冷冻干燥后即为当归粗多糖。
所述离心条件为转速4000r/min,离心时间为10min。
上述的当归多糖在制备预防或治疗辐射所致肠道屏障损伤药物中的应用。
上述的当归多糖在制备预防或治疗辐射所致血清DAO含量升高药物中的应用。
上述的当归多糖在制备预防或治疗辐射所致骨髓有核细胞含量降低药物中的应用。
上述的当归多糖在制备预防或治疗辐射所致骨髓白细胞含量降低药物中的应用。
上述的当归多糖在制备预防或治疗辐射所致肠道菌群失调药物中的应用。
本发明的有益效果在于:
药理实验结果显示:
当归多糖低、中、高剂量组大鼠血清DAO含量与模型组相比,均有显著统计学差异(P<0.008),提示当归多糖可以显著降低辐射引起的大鼠血清DAO含量升高,可显著减轻辐射引起的大鼠肠壁的损伤。
当归多糖低、中、高剂量组大鼠骨髓有核细胞、白细胞含量与模型组相比,均有显著统计学差异(P<0.008),提示当归多糖可以显著降低辐射引起的大鼠有核细胞、白细胞含量降低,可显著减轻辐射引起的大鼠造血功能的减退。
当归多糖低、中、高剂量组大鼠大便球杆比与模型组相比,均有显著统计学差异(P<0.008),提示当归多糖可以显著降低辐射引起的大鼠大便球杆比含量升高,可显著减轻辐射引起的大鼠肠道菌群失调。
附图说明
图1为空白组大鼠结肠组织的病理变化(HE,20×10)。
图2为模型组大鼠结肠组织的病理变化(HE,20×10)。
图3为低剂量组大鼠结肠组织的病理变化(HE,20×10)。
图4为中剂量组大鼠结肠组织的病理变化(HE,20×10)。
图5为高剂量组大鼠结肠组织的病理变化(HE,20×10)。
图6为空白组大鼠回肠组织的病理变化(HE,200×10)。
图7为模型组大鼠回肠组织的病理变化(HE,200×10)。
图8为低剂量组大鼠回肠组织的病理变化(HE,200×10)。
图9为中剂量组大鼠回肠组织的病理变化(HE,200×10)。
图10为高剂量组大鼠回肠组织的病理变化(HE,200×10)。
具体实施方式
下面结合具体实施例对本发明作更进一步的说明,以便本领域的技术人员更了解本发明,但并不因此限制本发明。
实施例1 当归多糖的制备
实验所用当归多糖由甘肃中药大学实验中心药理实验室用水提醇沉法制备。将自然风干后的新鲜当归10kg,用蒸馏水煮沸30min,当归与蒸馏水的料液比为1:30,提取液用无水乙醇调乙醇终浓度至80%,搅拌均匀后,4℃冷藏静置6h后离心,离心条件为转速4000r/min,离心时间为10min,沉淀用95%%乙醇去除色素直到无色为止,沉淀冷冻干燥后即为当归粗多糖。
实施例2 当归多糖预防SD大鼠电离辐射诱发肠道屏障损伤的药理实验
1分组与给药
清洁级雄性SD大鼠共40只,随机分为空白对照组、模型组,当归多糖63.56mg/kg组,当归多糖127.1mg/kg组,当归多糖254.2mg/kg组,每组8只动物。每组在辐照前7天和照射后3天给予相对应的灌胃液。空白对照组和模型组灌胃生理盐水,当归多糖低、中、高剂量组分别灌喂63.6 mg/kg、127.1 mg/kg和254.2 mg/kg当归多糖,连续灌胃7d后,用X射线6Gy分两次照射,照射后继续灌胃3天。
2动物辐照
除空白组外其余各组均用X线仪进行全身照射,辐照剂量率0.9226Gy/min,距离辐射源70cm高度的X射线仪进行背部辐射,第一天辐照剂量为3Gy,第二天以相同的方式进行腹部辐射,剂量也为3Gy,总剂量为6Gy。辐照后三天股动脉取血并处死大鼠检测相应的指标。
结果
3.1 一般情况观察结果
分别用辐射后第二天、第三天的体重减去辐射前一天的体重,再除以辐射前一天的体重,得出大鼠体质量指数。SD大鼠辐射后出现不同程度的体质量的变化结果见表1。
表1 各组大鼠体质量指数比较(`x±s)
组别 | n | 辐射后第二天 | 辐射后第三天(处死) |
空白组 | 8 | 0.030±0.014<sup>△</sup> | 0.048±0.022<sup>△</sup> |
模型组 | 7 | -0.051±0.011<sup>*</sup> | -0.104±0.032<sup>*</sup> |
低剂量组 | 7 | -0.049±0.011<sup>*</sup> | -0.090±0.012<sup>*</sup> |
中剂量组 | 7 | -0.039±0.020<sup>*</sup> | -0.071±0.031<sup>*</sup> |
高剂量组 | 6 | -0.038±0.016<sup>*</sup> | -0.103±0.062<sup>*</sup> |
与空白组比较,*P<0.008;与模型组比较,△P<0.008
3.2肠道屏障功能检测结果
3.2.1各组大鼠肠组织病理变化
回肠、结肠常规HE染色病理变化观察 动物回肠、结肠组织做苏木精-伊红(HE)染色,通过光学显微镜观察各组大鼠肠组织病理性形态学变化;观察每组大鼠肠道组织的病理损伤情况。结果显示:空白组大鼠肠组织结构完整,肠绒毛排列整齐。而辐射后的大鼠肠组织结构出现了不同程度的损伤,细胞核大,有异型性表现,腺体扩张明显,绒毛水肿,粘膜糜烂甚至坏死脱落,粘膜和粘膜下基层分离,平滑肌部分断裂。结果见图1-10。
3.2.2各组大鼠血清DAO含量变化
在大鼠处死时取血检测血清DAO。依据说明书操作步骤检测试剂与样本反应前后吸光度值,求出二者差值后再依据说明书给出公式计算血清DAO的数值。
表2 各组大鼠血清DAO含量比较(`x±s)
组别 | n | 血清DAO |
空白组 | 6 | 33.96±5.34△ |
模型组 | 6 | 82.62±9.19* |
低剂量组 | 6 | 56.26±6.93*△ |
中剂量组 | 6 | 50.30±9.64*△ |
高剂量组 | 6 | 50.94±9.19*△ |
与空白组比较,*P<0.008;与模型组比较,△P<0.008。
结果显示,当归多糖低、中、高剂量组大鼠血清DAO含量与模型组相比,均有显著统计学差异(P<0.008),提示当归多糖可以显著降低辐射引起的大鼠血清DAO含量升高,可显著减轻辐射引起的大鼠肠壁的损伤。
3.2.3各组大鼠骨髓有核细胞、白细胞计数变化
股动脉取血后颈椎脱臼处死大鼠,每组分离出大鼠的左股骨,用剪刀延股骨颈将股骨头剪掉,用10mL3%的冰醋酸通过5mL的注射器将骨髓全部冲出,并用移液器轻轻吹打,尽量使细胞吹散,用血球计数板计数有核细胞的数量(有核细胞数=(四个大格的细胞数/4)*10000)。
表3 各组大鼠骨髓有核细胞、白细胞计数比较(`x±s)
组别 | n | 骨髓有核细胞计数 | 白细胞计数 |
空白组 | 6 | 889.50±43.20<sup>△</sup> | 6.64±0.85<sup>△</sup> |
模型组 | 6 | 39.00±2.97<sup>*</sup> | 0.08±0.02<sup>*</sup> |
低剂量组 | 6 | 104.67±7.97<sup>*△</sup> | 1.54±0.18<sup>*△</sup> |
中剂量组 | 6 | 150.33±12.49<sup>*△</sup> | 1.63±0.19<sup>*△</sup> |
高剂量组 | 6 | 126.50±3.39<sup>*△</sup> | 1.48±0.24<sup>*△</sup> |
与空白组比较,*P<0.008;与模型组比较,△P<0.008。
结果显示,当归多糖低、中、高剂量组大鼠骨髓有核细胞、白细胞含量与模型组相比,均有显著统计学差异(P<0.008),提示当归多糖可以显著降低辐射引起的大鼠有核细胞、白细胞含量降低,可显著减轻辐射引起的大鼠造血功能的减退。
3.2.5 各组大鼠肠道菌群比值改为变化
处死前收集大鼠粪便,革兰氏染色后油镜镜检,拍摄照片,计数球菌及杆菌数量,观察菌群变化,计算球杆比。统计参考周庭银《临床微生物学诊断与图解(第3版)》标准。
表5 各组大鼠肠道菌群比较(`x±s)
组别 | n | 球杆比 | G<sup>+</sup>/G<sup>-</sup> |
空白组 | 6 | 0.48±0.24<sup>△</sup> | 1.25 ±0.341<sup>△</sup> |
模型组 | 6 | 2.48±0.45<sup>*</sup> | 0.82 ±0.55** |
低剂量组 | 6 | 1.10±0.22<sup>*△</sup> | 1.13 ±0.32*<sup>△</sup> |
中剂量组 | 6 | 1.15±0.35<sup>*△</sup> | 1.22 ±0.25*<sup>△</sup> |
高剂量组 | 6 | 1.04±0.36<sup>*△</sup> | 1.26 ±0.31*<sup>△</sup> |
与空白组比较,*P<0.008;与模型组比较,△P<0.008。
结果显示,当归多糖低、中、高剂量组大鼠大便球杆比、球菌革兰氏染色阴阳比与模型组相比,均有显著统计学差异(P<0.008),提示当归多糖可以显著降低辐射引起的大鼠大便球杆比含量升高,可显著减轻辐射引起的大鼠肠道菌群失调。
Claims (1)
1.当归多糖在制备治疗辐射所致血清DAO含量升高药物中的应用,其特征在于,所述当归多糖提取方法为:将自然风干后的新鲜当归,直接用蒸馏水煮沸30min,料液比为1∶30,提取液用无水乙醇调乙醇终浓度至80%,搅拌均匀后,4℃冷藏静置6h后离心,沉淀用95%乙醇去除色素直到无色为止,沉淀冷冻干燥后即为当归粗多糖;所述离心条件为转速4000r/min,离心时间为10min。
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